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NMDA-type glutamate receptors are ligand-gated ion channels that mediate a Ca2+-permeable component of excitatory neurotransmission in the central nervous system (CNS). They are expressed throughout the CNS and play key physiological roles in synaptic function, such as synaptic plasticity, learning, and memory. NMDA receptors are also implicated in the pathophysiology of several CNS disorders and more recently have been identified as a locus for disease-associated genomic variation. NMDA receptors exist as a diverse array of subtypes formed by variation in assembly of seven subunits (GluN1, GluN2A-D, and GluN3A-B) into tetrameric receptor complexes. These NMDA receptor subtypes show unique structural features that account for their distinct functional and pharmacological properties allowing precise tuning of their physiological roles. Here, we review the relationship between NMDA receptor structure and function with an emphasis on emerging atomic resolution structures, which begin to explain unique features of this receptor.

Introduction

The vast majority of the excitatory neurotransmission in the central nervous system (CNS) is mediated by vesicular release of glutamate, which activates both pre and postsynaptic G-protein–coupled metabotropic glutamate receptors and ionotropic glutamate receptors (iGluRs). iGluRs are ligand-gated cation channels that are divided into three major structurally distinct functional classes: the α-amino-3-hydroxy-5-methyl-4-isoxasolepropionic acid (AMPA) receptors, kainate receptors, and NMDA receptors (Traynelis et al., 2010; Fig. 1 A). The nomenclature for these functional classes was initially based on the activating agonist, and subsequent molecular cloning revealed cDNAs encoding multiple subunits within the three classes of iGluRs. An intriguing fourth class of iGluRs (GluD1-2) have structural resemblance to AMPA and kainate receptors but do not function as ion channels under normal circumstances (Yuzaki and Aricescu, 2017). Several unique properties distinguish NMDA receptors from other glutamate receptors, including voltage-dependent block by extracellular Mg2+, high permeability to Ca2+, and the requirement for binding of two coagonists, glutamate and glycine (or d-serine), for channel activation (Traynelis et al., 2010). These features have a profound impact on the physiological roles of NMDA receptors and have therefore been the topic of intense investigation.

Functional classes of iGluRs. (A) iGluRs are divided into AMPA, kainate, and NMDA receptors with multiple subunits cloned in each of these functional classes. (B) EPSCs from central synapses can be divided into fast AMPA or slow NMDA receptor–mediated components in the absence of Mg2+ using the AMPA receptor antagonist CNQX or the NMDA receptor antagonist AP5. The figure is adapted from Traynelis et al. (2010). (C) The relationships between NMDA receptor current response and membrane potential (i.e., holding potential) in the presence and absence of 100 µM extracellular Mg2+ reveal the voltage-dependent Mg2+ block, which is relieved as the membrane potential approaches 0 mV (i.e., with depolarization). Data are from Yi et al. (2018).

Functional classes of iGluRs. (A) iGluRs are divided into AMPA, kainate, and NMDA receptors with multiple subunits cloned in each of these functional classes. (B) EPSCs from central synapses can be divided into fast AMPA or slow NMDA receptor–mediated components in the absence of Mg2+ using the AMPA receptor antagonist CNQX or the NMDA receptor antagonist AP5. The figure is adapted from Traynelis et al. (2010). (C) The relationships between NMDA receptor current response and membrane potential (i.e., holding potential) in the presence and absence of 100 µM extracellular Mg2+ reveal the voltage-dependent Mg2+ block, which is relieved as the membrane potential approaches 0 mV (i.e., with depolarization). Data are from Yi et al. (2018).

At central synapses, glutamate release activates iGluRs that mediate an inward current and thereby depolarize the postsynaptic neurons. These excitatory postsynaptic currents (EPSCs) can be described primarily by two temporally distinct components corresponding to activation of AMPA and NMDA receptors. AMPA receptors mediate a synaptic current with rapid rise time and decay, whereas NMDA receptor activation mediates a current that activates more slowly with a time course that endures for tens to hundreds of milliseconds (Hestrin et al., 1990; Sah et al., 1990; Trussell et al., 1993; Geiger et al., 1997; Fig. 1 B). At rest, the NMDA receptor pore is strongly blocked in a voltage-dependent manner by extracellular Mg2+, but this block can be released by the depolarization that accompanies rapid activation of AMPA receptors, particularly when there is a series of closely spaced synaptic events (Fig. 1 C). Thus, the current mediated by NMDA receptors is dependent on both the membrane potential and frequency of synaptic release, rendering these receptors coincidence detectors that respond uniquely to simultaneous presynaptic release of glutamate and postsynaptic depolarization with a slow synaptic current that allows substantial influx of external Ca2+ into the dendritic spine (Bourne and Nicoll, 1993; Seeburg et al., 1995; Nevian and Sakmann, 2004). This increase in intracellular Ca2+ serves as a signal that leads to multiple changes in the postsynaptic neuron, including changes that ultimately produce either short-term or long-term changes in synaptic strength (Lau and Zukin, 2007; Holtmaat and Svoboda, 2009; Traynelis et al., 2010; Higley and Sabatini, 2012; Zorumski and Izumi, 2012; Paoletti et al., 2013; Volianskis et al., 2015). The nature of these changes (e.g., increased or decreased synaptic strength) depends on the frequency and duration of synaptic NMDA receptor activation (Citri and Malenka, 2008; Granger and Nicoll, 2013), thereby providing the brain with a mechanism for encoding information (Hunt and Castillo, 2012; Morris, 2013).

There is a rapidly increasing body of data from crystal or cryo-EM structures of intact NMDA receptors or individual domains, which provides a structural framework in which to consider biophysical properties of the receptors and allosteric modulation. In this review, we will focus on how emerging structural understanding has provided functional insight into key properties of the NMDA receptor that are relevant to its roles in the CNS.

When glutamate is released into the synaptic cleft, it reaches a high concentration (∼1.1 mM) for a brief duration of time, decaying with a time constant of ∼1.2 ms (Clements et al., 1992) as a result of diffusion and active removal of glutamate from the synaptic cleft by excitatory amino acid transporters (i.e., glutamate transporter; Divito and Underhill, 2014). In the synaptic cleft, glutamate will bind to AMPA (and/or kainate) and NMDA receptors, inducing the necessary conformational changes that trigger opening of the ion channel pore, a process referred to as gating. The NMDA receptor–mediated component of the EPSC continues to pass current for tens to hundreds of milliseconds after synaptic glutamate is removed (Lester et al., 1990), which is in part a reflection of agonist binding affinity but also because the receptor activation mechanism involves pregating steps as well as repeated transitions between glutamate-bound open and closed conformational states until glutamate eventually unbinds and the EPSC is terminated (Lester et al., 1990; Lester and Jahr, 1992; Erreger et al., 2005a; Zhang et al., 2008). The functional consequences of the gating reaction mechanism are strongly dependent on the identity of the GluN2 subunit (Monyer et al., 1992, 1994; Vicini et al., 1998; Wyllie et al., 1998). The four different GluN2 subunits thus create substantial diversity among NMDA receptors, and assembly of receptors that contain different GluN2 subunits with distinct properties allows tuning of the synaptic response time course and variation in parameters that control synaptic strength and plasticity. This diversity exerts many effects on neuronal function, circuit properties, and nervous system development.

Alternative splicing of exons 21 and 22 changes the amino acid composition of the intracellular GluN1 CTD, which interacts with PSD-95, calmodulin, and the neurofilament subunit NF-L (Traynelis et al., 2010). These proteins are involved in surface trafficking and anchoring of receptors at synaptic sites, and alternative splicing of exons 21 and 22 influences cell surface distribution of NMDA receptors (Scott et al., 2001, 2003; Mu et al., 2003; Wenthold et al., 2003). The CTD of GluN1 is a binding site for calmodulin (Ehlers et al., 1996; Iacobucci and Popescu, 2017b), as well as a target of kinases and phosphatases (Tingley et al., 1993, 1997). The relationships between functional roles and structural features of the residues encoded by GluN1 exon 21 and 22 are not yet fully understood.

Expression and functional properties of NMDA receptor subtypes determined by the GluN2 subunit. (A) Autoradiograms obtained by in situ hybridizations of oligonucleotide probes to parasagittal sections of rat brain at indicated postnatal (P) days reveal distinct regional and developmental expression of GluN2 subunits. Fig. 3 A is modified from Akazawa et al., 1994 with permission from the Journal of Comparative Neurology. (B) Whole-cell patch-clamp recordings of responses from recombinant diheteromeric NMDA receptor subtypes expressed in HEK293 cells. The receptors are activated by a brief application of glutamate (1 ms of 1 mM glutamate) indicated by the open tip current in the upper trace. Fig. 3 B is adapted from Vicini et al. (1998) with permission from the Journal of Neurophysiology. (C) Single-channel recordings of currents from outside-out membrane patches obtained from HEK293 cells expressing recombinant NMDA receptor subtypes. The single-channel recordings demonstrate distinct open probabilities and channel conductances depending on the GluN2 subunit in the diheteromeric NMDA receptor. Highlights of individual openings are shown on the left. Adapted from Yuan et al. (2008).

Expression and functional properties of NMDA receptor subtypes determined by the GluN2 subunit. (A) Autoradiograms obtained by in situ hybridizations of oligonucleotide probes to parasagittal sections of rat brain at indicated postnatal (P) days reveal distinct regional and developmental expression of GluN2 subunits. Fig. 3 A is modified from Akazawa et al., 1994 with permission from the Journal of Comparative Neurology. (B) Whole-cell patch-clamp recordings of responses from recombinant diheteromeric NMDA receptor subtypes expressed in HEK293 cells. The receptors are activated by a brief application of glutamate (1 ms of 1 mM glutamate) indicated by the open tip current in the upper trace. Fig. 3 B is adapted from Vicini et al. (1998) with permission from the Journal of Neurophysiology. (C) Single-channel recordings of currents from outside-out membrane patches obtained from HEK293 cells expressing recombinant NMDA receptor subtypes. The single-channel recordings demonstrate distinct open probabilities and channel conductances depending on the GluN2 subunit in the diheteromeric NMDA receptor. Highlights of individual openings are shown on the left. Adapted from Yuan et al. (2008).

NMDA receptor structure and function

All glutamate receptor subunits share a similar architecture that comprises four domains: a large extracellular ATD, a bilobed ABD, a pore-forming transmembrane domain (TMD), and an intracellular CTD (Fig. 4 A). The TMD is formed by three transmembrane helices (M1, M3, and M4) and a reentrant loop (M2). In iGluRs, the reentrant loop lines the intracellular portion of the ion channel pore, whereas elements of the third transmembrane segment (M3) form the extracellular region of the pore. Among NMDA receptor subtypes, the residues in the pore region, which influence ion permeation, are highly conserved. A key determinant of ion permeation, which controls divalent ion permeability and Mg2+ block, resides at the apex of the reentrant M2 loop and is often referred to as the Q/R/N site on the basis of amino acids at this position in AMPA, kainate, and NMDA receptors (Wollmuth, 2018).

Domain organization and ligand-binding sites in NMDA receptors. (A) The linear representation of the polypeptide chain illustrates the segments that form the four semiautonomous subunit domains shown in the cartoon, which are the extracellular ATD, the ABD, the TMD formed by three transmembrane helices (M1, M2, and M4) and a membrane reentrant loop (M2), and the intracellular CTD. The ABD is formed by two polypeptide segments (S1 and S2) that fold into a bilobed structure with an upper lobe (D1) and lower lobe (D2). The agonist-binding site is located in the cleft between the two lobes. (B) The crystal structure of the GluN1/2B NMDA receptor (Protein Data Bank accession no. 4PE5; Karakas and Furukawa, 2014) shows the subunit arrangement and the layered domain organization. The binding sites for agonists (and competitive antagonists) as well as predicted and known binding sites for PAMs and NAMs are highlighted. The figure is adapted from Hansen et al. (2017).

Domain organization and ligand-binding sites in NMDA receptors. (A) The linear representation of the polypeptide chain illustrates the segments that form the four semiautonomous subunit domains shown in the cartoon, which are the extracellular ATD, the ABD, the TMD formed by three transmembrane helices (M1, M2, and M4) and a membrane reentrant loop (M2), and the intracellular CTD. The ABD is formed by two polypeptide segments (S1 and S2) that fold into a bilobed structure with an upper lobe (D1) and lower lobe (D2). The agonist-binding site is located in the cleft between the two lobes. (B) The crystal structure of the GluN1/2B NMDA receptor (Protein Data Bank accession no. 4PE5; Karakas and Furukawa, 2014) shows the subunit arrangement and the layered domain organization. The binding sites for agonists (and competitive antagonists) as well as predicted and known binding sites for PAMs and NAMs are highlighted. The figure is adapted from Hansen et al. (2017).

The ABD is formed by the S1 and S2 segments of the polypeptide chain, which are separated by the M1, M2, and M3 segments. The ABDs form kidney-shaped bilobed structures that contain an upper lobe (D1) and a lower lobe (D2) with the agonist-binding site residing in the cleft between these two lobes (Fig. 4 A). The ABD structure, intra- and intersubunit interactions, and its influence on receptor function have been studied for more than two decades. More recently, crystallographic and cryo-EM data have provided the first glimpses of the domain organization of inactive and active GluN1/2B NMDA receptors, providing mechanistic hypotheses by which the different domains and their cognate ligands influence receptor function (Karakas and Furukawa, 2014; Lee et al., 2014; Tajima et al., 2016; Zhu et al., 2016; Regan et al., 2018; Song et al., 2018). We will consider each of these domains in more detail below.

The GluN2A ABD in complex with the GluN1 ABD provided the first structural information about a GluN1/GluN2 subunit interface within the NMDA receptor complex, in addition to the binding mode for glutamate and glycine between the two lobes (D1 and D2) of GluN2A and GluN1, respectively (Furukawa et al., 2005; Fig. 5 A). Multiple water molecules reside in close proximity to the agonists, and some form a hydrogen-bonding network that interacts with the ligand. The glycine-binding pocket in GluN1 is considerably smaller and more hydrophobic than the glutamate-binding pocket in GluN2 (Furukawa and Gouaux, 2003; Furukawa et al., 2005; Inanobe et al., 2005; Yao et al., 2008, 2013). Residues within the glutamate-binding pocket that make atomic contacts with agonists or competitive antagonists are mostly conserved in the GluN2 subunits, and it has therefore proven difficult to identify ligands that bind to this site with strong selectivity between the different NMDA receptor subtypes. However, recent crystallographic data have revealed the structural basis for binding of antagonists with modest selectivity (Lind et al., 2017; Romero-Hernandez and Furukawa, 2017). Selectivity in these cases is driven by space outside the conventional binding pocket that competitive antagonists can exploit in a GluN2-dependent manner (Fig. 5 B).

Crystal structures of NMDA receptor ABDs. (A) Structures of the soluble GluN1/2 ABD heterodimers reveal the subunit interface and back-to-back dimer arrangement of the ABDs. The structure shown here is for the GluN1/2A ABD heterodimer with bound glutamate and glycine shown as spheres (Protein Data Bank accession no. 5I57; Yi et al., 2016). The top view of the structure highlights sites I–III at the subunit interface. (B) Overlay of crystal structures of GluN1/2A ABD heterodimers in complex with glycine and either glutamate agonist (Protein Data Bank accession no. 5I57; Yi et al., 2016) or a competitive glutamate site antagonist (Protein Data Bank accession no. 5U8C; Romero-Hernandez and Furukawa, 2017). Activation of NMDA receptors requires agonist-induced ABD closure. Competitive antagonists bind the ABD without inducing domain closure, thereby preventing receptor activation. (C) Magnified views of the glutamate-binding site with bound GluN2A-preferring antagonists NVP-AAM077 (Protein Data Bank accession no. 5U8C; Romero-Hernandez and Furukawa, 2017) or ST3 (Protein Data Bank accession no. 5VII; Lind et al., 2017). Schild analyses demonstrated that NVP-AAM077 has 11-fold and ST3 has 15-fold preference for GluN1/2A over GluN1/2B receptors (data adapted from Lind et al., 2017). The crystal structures reveal a binding mode in which NVP-AAM077 and ST3 occupy a cavity that extends toward GluN1 at the subunit interface, and mutational analyses show that the GluN2A preference of these antagonists is primarily mediated by four nonconserved residues (Lys738, Tyr754, Ile755, and Thr758) that do not directly contact the ligand but are positioned within 12 Å of the glutamate-binding site. (D) Structure of the agonist-bound GluN1/2A ABD heterodimer with the NAM MPX-007 bound at site II in the subunit interface (Protein Data Bank accession no. 5I59; Yi et al., 2016). (E) Magnified views of site II in GluN1/2A ABD heterodimer with bound MPX-007 (NAM; Protein Data Bank accession no. 5I59; Yi et al., 2016) or PAM GNE-8324 (Protein Data Bank accession no. 5H8Q; Hackos et al., 2016). The overlay illustrates the distinct effects of NAM and PAM binding on Val783 in GluN2A and Tyr535 in GluN1. The GluN2A selectivity of the NAMs and PAMs binding at this modulatory site is mediated by Val783 in GluN2A, which is nonconserved among GluN2 subunits (Phe in GluN2B and Leu in GluN2C/GluN2D).

The residues at the heterodimer interface between the GluN1 and GluN2 ABDs modulate receptor function in several important ways. Three separate areas of contact between GluN1 and GluN2A can be seen in the ABD heterodimer crystal structures (referred to as sites I, II, and III; Furukawa et al., 2005; Fig. 5 A). Sites I and III consist of hydrophobic residues from both GluN1 and GluN2, and nonpolar interactions between these residues mediate ABD heterodimerization (Furukawa et al., 2005). The heterodimeric arrangement of GluN1 and GluN2A ABDs is similar to the homodimeric arrangement found in some AMPA and kainate receptors. In AMPA receptors, allosteric modulators such as cyclothiazide and aniracetam bind to sites equivalent to site I + III and site II, respectively, resulting in block of desensitization and slowing of deactivation speeds (Sun et al., 2002; Jin et al., 2005). The aromatic ring of Tyr535 in GluN1 has a positional overlap with that of aniracetam bound in AMPA receptors, therefore acting like a natural “tethered ligand” incorporated in the primary sequence (Furukawa et al., 2005). Consistently, mutations of Tyr535 in GluN1 alters deactivation time course of NMDA receptors, suggesting that the heterodimer interface can influence factors controlling deactivation, such as agonist dissociation or channel open time (Furukawa et al., 2005; Borschel et al., 2015). Recent crystallographic studies have shown that site II of the GluN1/2A ABD heterodimer contains the binding sites for both positive and negative allosteric modulators with strong selectivity for GluN2A (Hackos et al., 2016; Volgraf et al., 2016; Yi et al., 2016). Together, these results identify the heterodimer ABD interface as an important locus for modulation of NMDA receptor function (Fig. 5 C).

Structures of the GluN1-GluN2A ABD heterodimer in complex with various agonists, partial agonists, and antagonists have suggested a structural basis for their modes of action (Furukawa and Gouaux, 2003; Furukawa et al., 2005; Inanobe et al., 2005; Vance et al., 2011; Hansen et al., 2013; Yao et al., 2013; Jespersen et al., 2014). Binding of glycine and glutamate to GluN1 and GluN2 ABDs, respectively, produces a rapid ABD rearrangement that involves reduction of the angle between the D1 and D2 lobes, producing a clamshell-like closure of the bilobed domain (Fig. 5 B). This agonist-mediated ABD closure triggers formation of hydrogen bonds between residues from the upper and lower lobes, which are hypothesized to stabilize the agonist-bound ABD structure (Kalbaugh et al., 2004; Paganelli et al., 2013). The energy provided by agonist binding and ABD closure triggers the receptor to undergo a series of conformational changes that ultimately open the ion channel pore. Thus, ABD closure that results from agonist binding is the initial conformational change that ultimately triggers the process of ion channel gating. Binding of competitive antagonists, such as the glycine site antagonist DCKA and the glutamate site antagonist D-AP5, stabilizes an open cleft conformation that is incapable of triggering channel gating (Fig. 5 B).

The stabilization of the NMDA receptor ABDs in a closed cleft conformation by agonist binding and in an open cleft conformation by competitive antagonist binding is similar to that found for the AMPA and kainate receptor ABDs (Pøhlsgaard et al., 2011; Kumar and Mayer, 2013; Karakas et al., 2015). However, one notable difference exists. Multiple structures of AMPA receptor ABDs in complex with partial agonists show partial domain closure that correlates with their efficacy (Pøhlsgaard et al., 2011). In contrast, multiple structures of ABD with bound partial agonists, such as d-cycloserine, ACPC, and ACBC in GluN1 and NMDA and Pr-NHP5G in GluN2 show virtually identical degrees of domain closure compared with structures with full agonists (Inanobe et al., 2005; Vance et al., 2011; Hansen et al., 2013). However, these crystal structures capture only one conformation of the isolated ABDs, which may be influenced by the lack of interacting domains (ATD and TMD) and is further stabilized by contacts in the crystal lattice. This caveat to crystal structures of the isolated ABDs is highlighted by recent single-molecule FRET and molecular dynamics studies that provide insight into the dynamic behavior of the NMDA receptor ABDs (Yao et al., 2013; Dai et al., 2015; Dai and Zhou, 2015; Dolino et al., 2015, 2016). These studies suggest that the ABDs fluctuate between open and closed cleft conformations even in the absence of agonist (i.e., the apo state). However, binding of full agonist changes the energy landscape for ABD conformations to strongly favor a fully closed conformation, whereas binding of partial agonists is less efficient in changing this landscape, thereby enabling the ABD to adopt conformations with intermediate domain closure more frequently than full agonists. Hence, a conformational selection mechanism is likely to account for partial agonism in NMDA receptors despite the lack of crystallographic data showing intermediate domain closure for partial agonists.

Structures of intact tetrameric NMDA receptors

The first structures of intact NMDA receptors (GluN1/2B extracellular domains and TMDs) confirmed the hypothesized domain organization and showed that GluN1 and GluN2B subunits exist in an alternating pattern (i.e., 1-2-1-2) within the tetrameric assembly (Karakas and Furukawa, 2014; Lee et al., 2014; Fig. 6). These studies also confirmed that the NMDA receptor structure shares certain characteristics with AMPA and kainate receptors. First, the receptor subunits adopt a layered structure, with one layer formed by TMDs and two extracellular layers formed by ABD heterodimers and ATD heterodimers. Second, the TMDs have a quasi-fourfold symmetry, whereas the extracellular portion shows twofold symmetry between the two ABD heterodimers and ATD heterodimers in a dimer-of-dimer arrangement. Thus, there is a symmetry mismatch between the TMD layer and the extracellular layers of the receptor. Third, there is a remarkable subunit crossover between the ABD layer and the ATD layer (Fig. 6). In addition, the NMDA receptor has several unique structural features when compared with AMPA and kainate receptors (Karakas and Furukawa, 2014; Lee et al., 2014). For example, there are extensive contacts between the two GluN1/2 ABD heterodimers that are not present in AMPA and kainate receptor structures. These contacts may provide the structural basis of the GluN2 subunit dependence of glycine potency. In addition, the NMDA receptor ATDs show a different arrangement, leading to distinct subunit interfaces compared with AMPA and kainate receptors. Importantly, the ATDs form extensive contacts with the upper lobe of the ABD whereas the ATD–ABD interactions are minimal in AMPA and kainate receptors. These interactions give the NMDA receptor a more compact “hot air balloon–like” appearance, which is distinct from the more Y-shaped AMPA and kainate receptors. The ABD–ATD interactions also create a protein–protein interface at which modulators can bind (Khatri et al., 2014; Kaiser et al., 2018). A recent study also showed that the motif encoded by exon 5, which controls pH sensitivity, deactivation time course, and agonist potency, is also located at the ABD–ATD interface and modulates the ABD–ATD interaction (Regan et al., 2018). The structure of the NMDA receptor thus reveals unique intra- and interdomain contacts that provide a framework for understanding allosteric interactions between subunits, as well as allosteric modulation by small-molecule ligands.

Structure of the intact NMDA receptors. (A) Structure of the glycine- and glutamate-bound GluN1-1b/2B NMDA receptor without CTDs (Protein Data Bank accession no. 5FXI; Tajima et al., 2016). (B) The GluN1 (1) and GluN2 (2) subunits are arranged as a dimer of heterodimers at the ATD and ABD layers in a 1-2-1-2 fashion. Note that the heterodimer pairs are interchanged between the ATD and ABD layers (i.e., subunit crossover). In the TMD layer, the GluN1 and GluN2 subunits are arranged as a tetramer with pseudo-fourfold symmetry. (C) Comparison of the two major conformational states observed in the presence of glycine and glutamate by cryo-EM/single-particle analysis. Shown in spheres are the Cα of the gating ring residues, GluN1-1b Arg684 and GluN2B Glu658, which are adjacent to the pore-forming M3 transmembrane helices. In the nonactive (Protein Data Bank accession no. 5FXI; Tajima et al., 2016) and active (Protein Data Bank accession no. 5FXG; Tajima et al., 2016) conformations, the distances between the two GluN2B Glu658 Cα atoms are ∼29 Å and ∼45 Å, respectively, indicating that degrees of tension in the ABD–TMD loops are different.

Structure of the intact NMDA receptors. (A) Structure of the glycine- and glutamate-bound GluN1-1b/2B NMDA receptor without CTDs (Protein Data Bank accession no. 5FXI; Tajima et al., 2016). (B) The GluN1 (1) and GluN2 (2) subunits are arranged as a dimer of heterodimers at the ATD and ABD layers in a 1-2-1-2 fashion. Note that the heterodimer pairs are interchanged between the ATD and ABD layers (i.e., subunit crossover). In the TMD layer, the GluN1 and GluN2 subunits are arranged as a tetramer with pseudo-fourfold symmetry. (C) Comparison of the two major conformational states observed in the presence of glycine and glutamate by cryo-EM/single-particle analysis. Shown in spheres are the Cα of the gating ring residues, GluN1-1b Arg684 and GluN2B Glu658, which are adjacent to the pore-forming M3 transmembrane helices. In the nonactive (Protein Data Bank accession no. 5FXI; Tajima et al., 2016) and active (Protein Data Bank accession no. 5FXG; Tajima et al., 2016) conformations, the distances between the two GluN2B Glu658 Cα atoms are ∼29 Å and ∼45 Å, respectively, indicating that degrees of tension in the ABD–TMD loops are different.

Although the crystallographic structures of intact NMDA receptors advance our understanding of the structure–function relationship, they nevertheless capture only a low energy conformation among the many conformations that the NMDA receptor moves through en route to activation. In these crystal structures, glycine and glutamate were bound to GluN1 and GluN2B, respectively, and the GluN2B-selective negative allosteric modulator ifenprodil was bound to the interface between GluN1 and GluN2B ATDs (Karakas and Furukawa, 2014; Lee et al., 2014). The structures represent the agonist-bound, inhibited receptor with the ion channel closed. However, recent cryo-EM data have described multiple conformations in the extracellular region, providing the first dynamic pictures of NMDA receptor conformational changes and insight into the structural mechanism of receptor activation and allosteric modulation (Tajima et al., 2016; Zhu et al., 2016). Unfortunately, the TMDs for the active and antagonist-bound states are not well resolved in the cryo-EM structures, limiting mechanistic insights into gating and antagonism. However, in the active conformation, distances between the residues that are in proximity of the TMDs increase as much as ∼20 Å in the context of the heterotetramer (Fig. 6 C). This “dilation” of the gating ring likely generates sufficient tensions in the ABD–TMD linker for rearrangement of the helices that form the gate (Kazi et al., 2014; Twomey and Sobolevsky, 2018). This tension can lead to reorientation of the M3 helix in AMPA receptors as well as a kink at an alanine residue that appears to serve as a hinge. Whether or not gating of NMDA receptor ion channels involves similar conformational alterations of the ABD–TMD linker and the TMD demonstrated in the recent AMPA receptor structures (Twomey and Sobolevsky, 2018) remains to be seen, although the gating motifs are highly conserved. Nevertheless, given that the relative orientation of the ABDs and TMDs is distinct in NMDA receptors, structural data are required to evaluate whether these ideas transfer between AMPA and NMDA receptors.

Control of NMDA receptor function by the ATD

The ATD adopts a bilobed structure, which is unrelated to the ABD, with R1 and R2 referring to upper and lower lobes, respectively (Karakas et al., 2009, 2011). Furthermore, there is a unique dimer-of-dimer arrangement of the NMDA receptor ATDs compared with the ATDs in AMPA and kainate receptors (Karakas and Furukawa, 2014; Lee et al., 2014; Meyerson et al., 2014; Sobolevsky, 2015; Tajima et al., 2016; Zhu et al., 2016). This arrangement, which is revealed in crystal and cryo-EM structures of intact iGluRs, is characterized by a protein–protein interface formed by the upper R2 lobes from the GluN1 and GluN2 subunits, whereas the lower R1 lobes, which connect to the ABDs, are almost completely separated.

Ligand binding to the ATD

Crystal structures have demonstrated that GluN2B-selective negative allosteric modulators (NAMs), such as ifenprodil and Ro 25–6981, bind to a modulatory site located at the subunit interface between GluN1 and GluN2B ATDs (Karakas et al., 2011; Karakas and Furukawa, 2014; Lee et al., 2014; Stroebel et al., 2016). These crystal structures revealed that only one residue in this modulatory site is different between GluN2A and GluN2B subunits, but sensitivity to ifenprodil is not introduced by converting this or other residues in GluN2A to that in GluN2B (Karakas et al., 2011; Burger et al., 2012). This stems from the fact that the intersubunit arrangements in GluN1/2A and GluN1/2B ATD heterodimers are distinct from each other, as demonstrated in the recent crystal structure of the GluN1/2A ATD heterodimer (Romero-Hernandez et al., 2016). Specifically, the “pocket” in the GluN1/GluN2 subunit interface is ideally sized to accommodate ifenprodil analogues in GluN1/2B, whereas such a pocket is absent in GluN1/2A because of the different subunit arrangement characterized by a ∼10° rotation compared with GluN1/2B. Multiple lines of investigation, including cryo-EM structures of intact NMDA receptors, functional studies, and computational analyses, suggest that ifenprodil inhibition involves closure of the GluN2B ATD bilobes with accompanying changes in the arrangement of the GluN1/2B ATD heterodimers (Burger et al., 2012; Tajima et al., 2016), indicating that both clamshell conformation and subunit arrangement are coupled to function of the NMDA receptor ion channel.

Functional and structural studies have converged on a structural model for NMDA receptor modulation by Zn2+ and ifenprodil, where modulator binding regulates receptor function through rearrangement of the ATD layer and GluN2 ATD clamshell opening and closing (Sirrieh et al., 2013, 2015a). In GluN1/2B, opening of the ATD bilobes robustly alters inter-GluN1/GluN2 subunit arrangement within the ATD, which results in a ∼13° rotation between the GluN1/2B ABD dimers and dilation of the gating ring (Tajima et al., 2016). NAMs such as ifenprodil and zinc favor the closure of the bilobed GluN2B ATD thereby “locking” the subunit arrangement in a way that prevents dilation of the gating ring. Interestingly, the zinc-bound GluN2A ATD is ∼13° more open compared with the zinc-bound GluN2B ATD (Karakas et al., 2009; Romero-Hernandez et al., 2016). This may explain in part the observation that the extent of zinc inhibition is smaller in GluN2A than GluN2B (Rachline et al., 2005).

NMDA receptors containing GluN1 with exon 5 (e.g., the GluN1-1b splice variant) have reduced sensitivity to all three allosteric modulators (Zn2+, ifenprodil, and spermine; Traynelis et al., 1995; Mott et al., 1998; Yi et al., 2018). In recent cryo-EM structures, the 21 amino acids encoded by exon 5 are placed just above the GluN1-GluN2 ABD heterodimer interface between the ATD and ABD layers, positioned to influence allosteric interactions between GluN2 ATD clamshell motions and GluN1-GluN2 ABDs (Regan et al., 2018). Furthermore, GluN2C residues from both the ATD and ABD that influenced the activity of PYD-106, a GluN2C-selective positive allosteric modulator (PAM), have been identified and molecular modeling proposed a modulatory binding site located in a pocket at the ATD–ABD interface of GluN2C (Khatri et al., 2014; Kaiser et al., 2018). These studies all point to the ATD as the major structural determinant of GluN2-specific variation among NMDA receptor subtypes. For this reason, allosteric modulation of NMDA receptors by the ATD is intensely investigated, and drug discovery studies are poised to identify novel ATD ligands with therapeutic potential.

What sequence of events leads to M3 rearrangement? Agonist binding to the bilobed ABDs involves a clamshell closure around the ligands that must be the first step in a sequence of conformational changes that lead to gating. These are followed by multiple short-lived, intermediate conformations that precede a rapid transition from the closed to the open state of the ion channel, inferred by brief, kinetically distinguishable closed states in the single-channel record and the relatively slow time course for receptor activation by supersaturating agonist (Banke and Traynelis, 2003; Popescu et al., 2004; Auerbach and Zhou, 2005; Erreger et al., 2005a; Schorge et al., 2005; Kussius and Popescu, 2009; Fig. 7). However, there is poor understanding of the protein conformations that represent the rate limiting steps en route to channel opening. Moreover, the lifetimes of some of these intermediate conformations are brief, suggesting they are unlikely to be captured in crystal structures or cryo-EM studies, leaving functional experiments as the most feasible (yet imperfect) way to glean clues as to how these changes might control channel opening. Recent functional studies have built explicit models of channel activation in which specific conformations are hypothesized for each of the four subunits (Gibb et al., 2018). Moreover, work with disease-causing mutations identified in human patients has provided key insights into the elements that comprise the gating control mechanism. Residues in the region connecting the S1 segment of the ABD with the M1 transmembrane helix (i.e., the pre-M1 linker) are invariant in the healthy population, and a locus for disease-associated mutations in various neurological diseases (Ogden et al., 2017). In addition, the region connecting the S2 segment of the ABD with the M4 transmembrane helix (i.e., the pre-M4 linker) also appears to be implicated in patients with NMDA receptor missense mutations, and both the pre-M1 and pre-M4 linkers are close enough to be in contact with the conserved SYTANLAAF motif in the M3 helical bundle crossing. These three elements (pre-M1, SYTANLAAF, and pre-M4) appear positioned to form a gating control mechanism (Chen et al., 2017), and it is possible that kinetically distinct conformational states may be the result of rearrangements of this triad of interacting regions. Moreover, the different amino acid sequences for pre-M1 and pre-M4 that exist for GluN1 and GluN2 as well as different positions of these elements in relation to the gating ring could lead to distinct lifetimes for intermediate conformations that must be traversed before rapid pore dilation (Erreger et al., 2005a; Dravid et al., 2008; Kussius and Popescu, 2009; Amico-Ruvio and Popescu, 2010; Vance et al., 2012).

Single-channel recordings of NMDA receptor gating. Recording of receptor activation (i.e., channel gating or pore dilation) in an excised outside-out membrane patch containing a single GluN1/2B receptor exposed to 1 mM glutamate plus 30 µM glycine for 1 ms as indicated. In this example, receptor activation results in a characteristically long burst of channel openings and closings (duration 128 ms). Evaluation of closed periods within the GluN1/2B activation suggests that two kinetically distinct pregating steps exist (i.e., fast and slow steps; see Fig. 8 D for model). Some (but not all) closures within the activation will reflect reversal of pore dilation, reversal of a single pregating step, followed by forward movement back through the pregating step and pore dilation. Two possible closures that might reflect the slow and fast pregating components are highlighted in red. Data are from Banke and Traynelis (2003).

Single-channel recordings of NMDA receptor gating. Recording of receptor activation (i.e., channel gating or pore dilation) in an excised outside-out membrane patch containing a single GluN1/2B receptor exposed to 1 mM glutamate plus 30 µM glycine for 1 ms as indicated. In this example, receptor activation results in a characteristically long burst of channel openings and closings (duration 128 ms). Evaluation of closed periods within the GluN1/2B activation suggests that two kinetically distinct pregating steps exist (i.e., fast and slow steps; see Fig. 8 D for model). Some (but not all) closures within the activation will reflect reversal of pore dilation, reversal of a single pregating step, followed by forward movement back through the pregating step and pore dilation. Two possible closures that might reflect the slow and fast pregating components are highlighted in red. Data are from Banke and Traynelis (2003).

Kinetic models for NMDA receptor activation

The sequence of protein conformational changes that trigger channel gating can be described as reaction schemes (i.e., kinetic models) with agonist binding steps and transitions between different conformational states of the receptor (Fig. 8). The first widely applied kinetic model for NMDA receptor gating was solely designed to account for the time course of the macroscopic current response and consisted of two identical but independent glutamate binding steps, one desensitized state, one closed state, and one open state (Lester and Jahr, 1992). This simple kinetic model appeared to effectively capture key features of macroscopic NMDA receptor responses but was not intended to describe the complexity observed in single-channel recordings (Ascher et al., 1988; Howe et al., 1991; Traynelis and Cull-Candy, 1991; Gibb and Colquhoun, 1992). Furthermore, the usefulness of the Lester and Jahr model was limited by the lack of glycine-binding steps required for receptor activation. Kinetic models that account for both glutamate- and glycine-binding steps as well as intersubunit interactions between the glutamate and glycine ABDs have also been developed (Benveniste et al., 1990), and these models could capture additional features of the time course of NMDA receptors, including glycine-dependent desensitization (see below).

Application of a gating reaction mechanism of NMDA receptors. (A) Individual responses from a recombinant GluN1/2B channel in an excised outside-out patch activated by 1 ms application of maximally effective glutamate and glycine (indicated by the gray vertical bar and the open tip recording above the channel recordings). The patch contained a single active channel, which allowed analysis of the variable delay before channel opening. NMDA receptors bind agonist rapidly and subsequently open after a multimillisecond delay that reflects transition through kinetically distinct protein conformations before pore dilation (i.e., channel gating). Note that although application of maximal glutamate and glycine always produces a binding event, not all binding events lead to channel opening. Reproduced from Erreger et al. (2005a). (B) The cumulative plot of latency to opening after application of 1 mM glutamate for 1 ms. (C) The average of all individual recordings of single activations produced a macroscopic waveform with a characteristic rise time. (D) Evaluation of closed periods within the GluN1/2B activation suggested a model where two pregating steps can occur in any order and explosive opening of the pore, which occurs faster than the resolution of the recordings, is assumed to happen instantaneously once both pregating steps have been traversed. (E) Simulation of a single activation for a GluN1/2B channel (using the model in D) illustrates how brief gaps can contain information about forward rates for the fast kinetically distinct pregating step. The color above the simulation indicates occupancy in the corresponding closed state of the model in D. The slow step often reverses again through the fast state (green) before reopening.

Application of a gating reaction mechanism of NMDA receptors. (A) Individual responses from a recombinant GluN1/2B channel in an excised outside-out patch activated by 1 ms application of maximally effective glutamate and glycine (indicated by the gray vertical bar and the open tip recording above the channel recordings). The patch contained a single active channel, which allowed analysis of the variable delay before channel opening. NMDA receptors bind agonist rapidly and subsequently open after a multimillisecond delay that reflects transition through kinetically distinct protein conformations before pore dilation (i.e., channel gating). Note that although application of maximal glutamate and glycine always produces a binding event, not all binding events lead to channel opening. Reproduced from Erreger et al. (2005a). (B) The cumulative plot of latency to opening after application of 1 mM glutamate for 1 ms. (C) The average of all individual recordings of single activations produced a macroscopic waveform with a characteristic rise time. (D) Evaluation of closed periods within the GluN1/2B activation suggested a model where two pregating steps can occur in any order and explosive opening of the pore, which occurs faster than the resolution of the recordings, is assumed to happen instantaneously once both pregating steps have been traversed. (E) Simulation of a single activation for a GluN1/2B channel (using the model in D) illustrates how brief gaps can contain information about forward rates for the fast kinetically distinct pregating step. The color above the simulation indicates occupancy in the corresponding closed state of the model in D. The slow step often reverses again through the fast state (green) before reopening.

All these kinetic models for NMDA receptor gating that faithfully describe both macroscopic responses and single-channel data require both multiple pregating steps and multiple open states. The interpretation of this observation is that ion channel opening in NMDA receptors is not directly coupled to agonist-induced ABD closure; instead, the receptor must proceed through a sequence of conformational changes that couple agonist binding to ion channel gating.

Structural determinants of ion permeation and channel block

The ion channel pore in NMDA receptors can be divided into the intracellular and extracellular vestibules separated by a narrow constriction (Fig. 9). The narrow restriction resides approximately halfway across the membrane at the apex of the membrane reentrant loop M2 (i.e., the Q/R/N site) and is often referred to as the selectivity filter because of its role as key determinant of Ca2+ permeability, single-channel conductance, and channel block (Wollmuth and Sobolevsky, 2004; Traynelis et al., 2010; Glasgow et al., 2015). The residue at the Q/R/N site is asparagine (N) in both GluN1 and GluN2, but the contribution of this residue to ion permeation is asymmetric between GluN1 and GluN2 subunits (Burnashev et al., 1992; Wollmuth et al., 1996, 1998; Sobolevsky et al., 2002b). This is because the narrow constriction is formed by the Q/R/N site asparagine in GluN1 but by the asparagine residue adjacent to the Q/R/N site (i.e., Q/R/N +1 site) in GluN2. The asymmetric contribution by GluN1 and GluN2 is revealed by substitutions of the Q/R/N site residue in GluN2 that have weak effects on Ca2+ permeability and dramatically reduce Mg2+ block, whereas the same substitutions of the Q/R/N site residue in GluN1 dramatically reduce Ca2+ permeability and have weak effects on Mg2+ block (Burnashev et al., 1992; Wollmuth et al., 1998). However, mutations at the Q/R/N +1 site in GluN2 strongly reduce Mg2+ block (Wollmuth et al., 1998). Functional data therefore suggest a structural asymmetry, where the apexes of M2 in GluN1 and GluN2 are slightly staggered (Sobolevsky et al., 2002b). Diheteromeric GluN1/3 receptors have glycine/arginine residues at the Q/R/N and Q/R/N +1 and show both markedly reduced Ca2+-permeability and Mg2+-block compared with GluN1/2 receptors (Cavara and Hollmann, 2008; Henson et al., 2010; Low and Wee, 2010; Pachernegg et al., 2012; Kehoe et al., 2013). Recent data describing the pore of the AMPA receptor in the open state reinforce the idea that the apex of the reentrant loops form a constriction that impacts ion permeation (Twomey et al., 2017). The structural basis for this functional asymmetry will require high-resolution images of the NMDA receptor in the open state.

General pore structure of NMDA receptors. (A) Pore-lining elements contributed by the GluN1 subunit (blue; Protein Data Bank accession no. 5UN1; Song et al., 2018). The M3 transmembrane segment lines the extracellular part of the permeation pathway, whereas the M2 pore loop lines the intracellular part with the N site asparagine (red circle) positioned at the tip of the M2 pore loop. The channel is in the closed conformation. (B) The narrow constriction is formed by nonhomologous asparagine residues, the GluN1 N site and the GluN2 N+1 site (Wollmuth et al., 1996; Song et al., 2018). The GluN2B subunit is colored orange. For both GluN1 and GluN2, the N site asparagine residue is positioned at the tip of the M2 loop.

General pore structure of NMDA receptors. (A) Pore-lining elements contributed by the GluN1 subunit (blue; Protein Data Bank accession no. 5UN1; Song et al., 2018). The M3 transmembrane segment lines the extracellular part of the permeation pathway, whereas the M2 pore loop lines the intracellular part with the N site asparagine (red circle) positioned at the tip of the M2 pore loop. The channel is in the closed conformation. (B) The narrow constriction is formed by nonhomologous asparagine residues, the GluN1 N site and the GluN2 N+1 site (Wollmuth et al., 1996; Song et al., 2018). The GluN2B subunit is colored orange. For both GluN1 and GluN2, the N site asparagine residue is positioned at the tip of the M2 loop.

Determinants of ion permeation

NMDA receptor ion channels are permeable to the physiologically relevant Ca2+, Na+, and K+ ions. The different NMDA receptor subtypes display similar permeability to Na+ and K+ ions (PK/PNa = 1.14) but are more permeable to Ca2+ relative to monovalent ions (PCa/PX = 1.8–4.5), with variation in Ca2+ permeability that depends on the GluN2 subunit (Burnashev et al., 1995; Schneggenburger, 1996, 1998; Sharma and Stevens, 1996; Jatzke et al., 2002). However, NMDA receptors also exhibit block by external Ca2+, despite being highly Ca2+ permeable, which can be observed as a reduction in channel conductance in single-channel data (Premkumar and Auerbach, 1996; Wyllie et al., 1996; Premkumar et al., 1997; Dravid et al., 2008). The concurrent block by Ca2+ and the high Ca2+ permeability are not incompatible properties but are expected if multiple Ca2+-binding sites are located in the ion channel pore of NMDA receptors (Premkumar and Auerbach, 1996; Sharma and Stevens, 1996). Studies have suggested that one Ca2+-binding site is located at the Q/R/N site, whereas another, more external Ca2+-binding site could be formed by a cluster of charged DRPEER residues in GluN1 (Watanabe et al., 2002; Karakas and Furukawa, 2014). The external Ca2+-binding site is located at the external entrance to the ion channel above the transmembrane helix M3 of GluN1. Although structural elements of Ca2+ permeation in GluN1/N2 subunits have been identified, the mechanism of Ca2+ permeation remains unknown.

Determinants of channel block

GluN1/2A and GluN1/2B channels are more strongly blocked by extracellular Mg2+ than GluN1/2C and GluN1/2D channels (Monyer et al., 1994; Kuner and Schoepfer, 1996; Qian et al., 2005; Clarke and Johnson, 2006; Siegler Retchless et al., 2012). This channel block is highly dependent on the membrane potential (i.e., voltage dependent), and the IC50 values (the concentrations that produce half-maximal inhibition) for block by external Mg2+ are 2 µM, 2 µM, 14 µM, and 10 µM for GluN1/2A, GluN1/2B, GluN1/2C, and GluN1/2D, respectively, at a holding potential of −100 mV (Kuner and Schoepfer, 1996). The dependency of Mg2+ block on the GluN2 subunit is influenced by multiple structural elements, but a main determinant appears to be a single residue at the S/L site located in the M3 transmembrane helix (Siegler Retchless et al., 2012). The residue at the S/L site, which is a serine in GluN2A/B and a leucine in GluN2C/D, is not lining the ion channel pore but has been suggested to interact with tryptophan residues in the membrane reentrant loop M2 of GluN1 (Siegler Retchless et al., 2012). This interaction between GluN1 and the GluN2 S/L site also appears to be a key determinant of GluN2 subunit–specific variation in channel conductance and Ca2+ permeability (Siegler Retchless et al., 2012). Although important insight into the structural mechanism of GluN2 subunit–dependent control of Mg2+ block is still missing, it is possible that structural elements, including the GluN2 S/L site, govern Mg2+ block by influencing binding sites for permeant ions in the channel pore (Antonov and Johnson, 1999; Zhu and Auerbach, 2001a,b; Qian et al., 2002; Qian and Johnson, 2006).

In general, the open channel blockers are considered nonselective among NMDA receptor subtypes (Dravid et al., 2007), but some channel blockers, such as ketamine and memantine, display 5- to 10-fold preference for GluN2C/D-containing receptors over GluN2A/B-containing receptors in the presence of 1 mM extracellular Mg2+ (i.e., under physiological conditions; Kotermanski and Johnson, 2009). NMDA receptor channel blockers have robust neuroprotective effects in animal models of CNS disorders that involve excessive NMDA receptor activation, such as stroke, epilepsy, and traumatic brain injury. However, clinical trials have not been successful because of dose-limiting side effects, patient heterogeneity, and a narrow temporal window for intervention that could have confounded interpretation (Ikonomidou and Turski, 2002; Farin and Marshall, 2004; Muir, 2006; see also Table S2 in Yuan et al., 2015). NMDA receptor channel blockers that bind with high affinity, such as ketamine and PCP, are typically dissociative anesthetics, and their clinical use is limited by psychomimetic side effects. Nonetheless, there is an intense interest in use of ketamine or similar molecules for the treatment of major depressive disorder because of several promising clinical trials in recent years based on the discovery of antidepressant effects for NMDA receptor antagonists (Niciu et al., 2014; Abdallah et al., 2015; Zanos et al., 2018).

Channel blockers such as memantine, which is approved in the treatment of moderate to severe Alzheimer’s disease, have lower affinity than ketamine and PCP and show faster blocking/unblocking kinetics (Parsons et al., 1993). These kinetic properties have been suggested to contribute to an improved therapeutic index with respect to psychomimetic effects, perhaps because of reduced channel block during normal synaptic transmission (Chen and Lipton, 2006), although the mechanism by which memantine may contribute to a symptomatic benefit in Alzheimer’s disease is not well understood. Interestingly, Glasgow et al. (2017) have proposed that memantine stabilizes occupancy of a desensitized state of GluN1/2A receptors, whereas ketamine reduces occupancy of a GluN1/2B desensitized state (Glasgow et al., 2017). Thus, the affinity of these blockers for their binding site in the channel may be allosterically affected by the conformational changes in the receptor protein associated with desensitization. Given the prevalence of triheteromeric GluN1/2A/2B receptors in the brain, it will be important to evaluate memantine and ketamine block at these triheteromeric receptors.

Endogenous mechanisms of functional modulation

NMDA receptors are complex macromolecular membrane-bound protein complexes, and their functional properties and membrane trafficking can be altered by extracellular ions, phosphorylation, and intracellular binding proteins. Additionally, the differences between various diheteromeric and triheteromeric NMDA receptor subtypes create selective actions of many of these types of modulation. Here, we will describe various forms of endogenous regulation of NMDA receptor function.

Modulation by protons

Extracellular protons inhibit NMDA receptor function with an IC50 of ∼50 nM, corresponding to a pH of ∼7.3 (Giffard et al., 1990; Traynelis and Cull-Candy, 1990, 1991; Vyklický et al., 1990). Thus, neuronal NMDA receptors are tonically inhibited by protons at physiological pH and are therefore poised to respond to small changes in extracellular pH that can occur under physiological conditions caused by release of protons from acidic synaptic vesicles or movement of protons across the plasma membrane by pumps (Chesler, 2003). Furthermore, pathological conditions, including seizure and ischemia, produce extracellular acidification, which can decrease pH to levels that strongly inhibit NMDA receptor function (Chesler, 2003).

The sensitivity of the NMDA receptors to inhibition by extracellular protons depends on the GluN2 subunit (Traynelis et al., 1995), with GluN2A-, GluN2B-, and GluN2D-containing NMDA receptors showing proton IC50 values near physiological pH (7.0–7.4). In contrast, GluN2C-containing receptors are less sensitive to changes in pH, with an IC50 value near pH 6.0 (Traynelis et al., 1995; Low et al., 2003). NMDA receptors that include GluN1 subunits containing the alternatively spliced exon 5 in the ATD (i.e., GluN1-1b) are notably less sensitive to protons (Traynelis et al., 1995). Proton inhibition is voltage independent, and without effect on glutamate potency; low pH produces modest shifts in the glycine potency (Tang et al., 1990; Traynelis and Cull-Candy, 1990, 1991; Traynelis et al., 1995). The structural determinants underlying proton inhibition are unknown, although mutations at the ABD interface, linkers to pore-forming elements, and within the M2 reentrant loop can all influence pH sensitivity (Low et al., 2003; Gielen et al., 2008). This suggests that NMDA receptor gating is tightly coupled to proton inhibition of the receptor. This idea is consistent with the observation that channel blockers appear to sense the protonation state of the receptor (Dravid et al., 2007).

The affinity for Zn2+ at the GluN2A ATD is high enough such that Zn2+ contamination in physiological levels of salts can produce significant inhibition (Paoletti et al., 1997). The effects of contaminant Zn2+ in functional experiments can be removed by inclusion of even low concentrations of divalent ion chelators, such as 10 µM EDTA. The high affinity of such chelators for Zn2+ means that even low micromolar levels of chelator will bind virtually all of the nanomolar-contaminating Zn2+ ions but exert minimal effects on millimolar concentrations of Ca2+ or Mg2+ (Anderson et al., 2015).

Positive and negative allosteric modulation by neurosteroids

Several endogenous neurosteroids positively and negatively modulate NMDA receptor activity (Traynelis et al., 2010), although the actions of these lipophilic molecules are complex. For instance, pregnenolone sulfate has dual actions on NMDA receptor responses, having both inhibitory and potentiating activity over a wide range of potencies (Horak et al., 2006). The potentiating actions of pregnenolone sulfate are most prominent when applied before receptor activation, whereas inhibitory actions arise when applied continuously (Horak et al., 2006). The dual actions of pregnenolone sulfate lead to divergent effects depending on the GluN2 subunit; when applied during steady-state NMDA receptor responses, GluN1/2A and GluN1/2B are potentiated, whereas GluN1/2C and GluN1/2D are inhibited.

Other neurosteroid analogues, such as pregnanolone sulfate, inhibit all NMDA receptor subtypes (i.e., they are pan-inhibitors) in a use-dependent manner through actions that involve the extracellular portion of the conserved M3 SYTANLAAF motif (Malayev et al., 2002). For GluN2A, pregnanolone sulfate has been proposed to increase the occupancy of a desensitized state (Kussius et al., 2009). In contrast, 24(S)-hydroxycholesterol and related analogues appear to be pan-potentiators, whereas 25(S)-hydroxycholesterol may antagonize actions of endogenous 24(S)-hydroxycholesterol (Paul et al., 2013; Linsenbardt et al., 2014). Some of these neurosteroids and analogues have been shown to exhibit agonist dependency, although this property is difficult to assess, because neurosteroids can have distinct actions on NMDA receptors, dependent on the timing of modulator application (i.e., see pregnenolone sulfate actions above). Additionally, steroid derivatives may partition into the membrane en route to their active site, which will alter the concentration–response relationship of their actions (Borovska et al., 2012; Vyklicky et al., 2015). A recent study reported that cholesterol modulates NMDA receptor function and its removal inhibited receptor activity (Korinek et al., 2015), suggesting that the membrane environment influences NMDA receptor activity and may be an important determinate of neurosteroid action. Although a clear binding site has not been resolved, it seems likely that neurosteroid derivatives interact directly with the receptor rather than simply alter membrane fluidity. A subset of neurosteroid inhibitors also have voltage-dependent actions, suggesting that they may inhibit NMDA receptors through blocking the channel (Vyklicky et al., 2015). These findings highlight the complexity associated with neurosteroid activity, and more work is required to delineate the mechanism of action of these compounds.

Desensitization of NMDA receptors

The process of desensitization is broadly defined as a decrease in a response in the continued presence of a stimulus. NMDA receptors exhibit several forms of desensitization, which can be distinguished on the basis of time course and mechanism, including glycine-, Zn2+-, and Ca2+-dependent desensitization. Most ligand-gated channels can desensitize in the continued presence of agonist by a mechanism thought to involve a conformational change to a stable and long-lived agonist-bound closed state. NMDA receptors can also desensitize in the continued presence of glutamate and glycine, presumably by this same mechanism, in a manner that is independent of glycine, Zn2+, and Ca2+. NMDA receptors exhibit only weak desensitization compared with the relatively strong desensitization of AMPA and kainate receptors. However, this desensitization is sensitive to intracellular dialysis, being more prominent in excised outside-out membrane patches (Sather et al., 1990, 1992), and is perturbed by mutations in a wide range of domains, including the conserved M3 gating motif, the pre-M1 linker region, the ion channel pore, the ABD, and the TMD–ABD interface (Chen et al., 2004; Hu and Zheng, 2005; Alsaloum et al., 2016).

Glycine-dependent NMDA receptor desensitization

Glycine-dependent NMDA receptor desensitization is present only in subsaturating glycine concentrations (Mayer et al., 1989) and occurs as a result of a negative allosteric interaction between the glutamate- and glycine-binding sites, such that the binding of glutamate decreases the glycine affinity and vice versa (Benveniste et al., 1990; Lester et al., 1993). Thus, when glutamate binds to GluN2 in the absence of high concentrations of glycine, the current will initially rise to a peak and then decline to a new equilibrium as glycine unbinds from the receptor after the allosteric reduction in glycine affinity. The time course for the desensitization is dictated by glycine unbinding (time constant ∼0.3 s) and is temporally close to the synaptic NMDA receptor time course, raising the possibility that glycine-dependent desensitization could impact synaptic signaling when glycine is subsaturating (Berger et al., 1998). Recent structural data for NMDA receptors provide plausible models for the negative allosteric coupling between glutamate- and glycine-binding sites, given their close proximity (Karakas and Furukawa, 2014; Lee et al., 2014; Tajima et al., 2016; Zhu et al., 2016). However, the structural features that enable glycine-dependent desensitization remain poorly understood.

Conclusion

Recent discoveries from genetic analyses that link NMDA receptors to specific disease conditions, the emerging evidence of the antidepressant effects of NMDA receptor antagonists, and accelerating identification of new subunit-selective modulators have reinvigorated the long-standing interest in NMDA receptors as therapeutic targets. The field now seems poised to achieve new levels of understanding of the functional roles of NMDA receptors in physiology and disease. The rapidly increasing volume of crystallographic and cryo-EM data has created an opportunity to view function through structure, which will inevitably improve our insight to the mechanisms by which agonist binding is linked to channel gating and how different NMDA receptor subunits contribute to the conformational changes that occur during gating and allosteric modulation. These new developments can lead novel perspectives in the NMDA receptor field and create exciting opportunities to study the physiological roles of different diheteromeric and triheteromeric NMDA receptor subtypes in distinct subcellular locations and neuronal populations. These converging advances in NMDA receptor pharmacology, mechanistic understanding of receptor function, and the basis of CNS diseases involving NMDA receptor dysfunction are already catalyzing the development of new therapeutic strategies.

Acknowledgments

This work was supported by grants from National Institutes of Health to S.F. Traynelis (NS036654 and NS065371), K.B. Hansen (GM103546 and NS097536), and L.P. Wollmuth (NS088479).

S.F. Traynelis is a consultant for Janssen Pharmaceuticals, Inc., the principal investigator on a research grant awarded to Emory University from Janssen Pharmaceuticals, a member of the Scientific Advisory Board for Sage Therapeutics, and co-founder of NeurOp, Inc. K.B. Hansen is the principal investigator on a research grant awarded to University of Montana from Janssen Pharmaceuticals. The other authors declare no competing financial interests.

Author contributions: All authors wrote the manuscript.

Lesley C. Anson served as editor.

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